Compositions comprising anti-cd38 antibodies and carfilzomib

ABSTRACT

Disclosed herein are compositions and kits which comprise anti-CD38 antibodies and carfilzomib compounds. Also disclosed are methods for treating cancers, such as multiple myeloma, in subjects with the compositions and kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 61/778,540,filed 13 Mar. 2013, and U.S. Application No. 61/808,381, filed 4 Apr.2013, all of which are herein incorporated by reference in theirentirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“20140313_(—)034543_(—)002WO1_seq” which is 56.7 kb in size was createdon 13 Mar. 2014 and electronically submitted via EFS-Web herewith theapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the present invention relates to anti-CD38 antibodies,carfilzomib, and cancer treatments.

2. Description of the Related Art

Multiple myeloma (MM) is a B cell malignancy. In MM, abnormal plasmacells accumulate in the bone marrow where they interfere with theproduction of normal cells. Current therapy of MM includesadministration of proteasome inhibitors such as bortezomib andcarfilzomib, immunomodulatory drugs such as lenalidomide andthalidomide, and chemotherapy such as melphalan and prednisone. Whilethese agents have improved survival in multiple myeloma, invariablyresistance becomes problematic and patients succumb from their illness.Multiple myeloma thus remains ultimately fatal, with a median survivalof approximately 3 to 5 years only.

CD38 is expressed on malignant plasma cells. CD38 is a 45 kD type IItransmembrane glycolprotein with a long C-terminal extracellular domainand a short N-terminal cytoplasmic domain. The CD38 protein is abifunctional ectoenzyme that can catalyze the conversion of NAD⁺ intocyclic ADP-ribose (cADPR) and also hydrolyze cADPR into ADP-ribose. CD38is up-regulated and has been implicated in many hematopoieticmalignancies.

Thus, some proposed MM treatments include the administration ofanti-CD38 antibodies. See, for example, WO 2012/041800; de Weers et al.(2011) J Immunol 186:1840-1848; and Van der Veer et al. (2011)Haematologica 96(2):284-290. Unfortunately, like various drugs andchemotherapies, not all antibodies are the same and not all antibodiesagainst the same antigen exhibit the same activities.

There is thus a need for new and efficacious treatments for extendingsurvival and improving outcome of treatments of multiple myeloma, andmore generally of blood cancers.

DESCRIPTION OF THE DRAWINGS

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are intended toprovide further explanation of the invention as claimed. Theaccompanying drawings are included to provide a further understanding ofthe invention and are incorporated in and constitute part of thisspecification, illustrate several embodiments of the invention, andtogether with the description serve to explain the principles of theinvention.

This invention is further understood by reference to the drawingswherein:

FIG. 1A shows the growth rate of tumors in xenograft models implantedwith NCI-H929 cells (H929 models).

FIG. 1B shows the growth rate of tumors in xenograft models implantedwith RPMI 8226 cells (RPMI models).

FIG. 2A shows the tumor volume of tumors in RPMI models after treatmentwith the indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 2B shows the body weight of the RPMI models after treatment withthe indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 3A shows the tumor volume of tumors in H929 models after treatmentwith the indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 3B shows the body weight of the H929 models after treatment withthe indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 4A shows the tumor volume of tumors in H929 models after treatmentwith the indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 4B shows the body weight of the H929 models after treatment withthe indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 5A shows the tumor volume of tumors in H929 models after treatmentwith the indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 5B shows the body weight of the H929 models after treatment withthe indicated dose of hu38SB19 at the indicated times (arrows).

FIG. 6A shows the tumor volume of tumors in H929 models after treatmentwith the indicated dose of carfilzomib at the indicated times (arrows).

FIG. 6B shows the body weight of the H929 models after treatment withthe indicated dose of carfilzomib at the indicated times (arrows).

FIG. 7A shows the tumor volume of tumors in RPMI models after treatmentwith the indicated dose of carfilzomib at the indicated times (arrows).

FIG. 7B shows the body weight of the RPMI models after treatment withthe indicated dose of carfilzomib at the indicated times (arrows).

FIG. 8A shows the tumor volume of tumors in H929 models after treatmentwith the indicated dose of hu38SB19 at the indicated times (top arrows)and the indicated dose of carfilzomib at the indicated times (bottomarrows).

FIG. 8B shows the body weight of the H929 models after treatment withthe indicated dose of hu38SB19 at the indicated times (top arrows) andthe indicated dose of carfilzomib at the indicated times (bottomarrows).

FIG. 9A is a graph showing the mean wet tumor weights of the H929 modelsafter the indicated treatment with carfilzomib and/or hu38SB19 (mAb).

FIG. 9B is a graph showing the median wet tumor weights of the H929models after the indicated treatment with carfilzomib and/or hu38SB19(mAb).

FIG. 10A shows the tumor volume of tumors in RPMI-8226 models aftertreatment with the indicated dose of hu38SB19 at the indicated times(top arrows) and the indicated dose of carfilzomib at the indicatedtimes (bottom arrows).

FIG. 10B shows the body weight of the RPMI-8226 models after treatmentwith the indicated dose of hu38SB19 at the indicated times (top arrows)and the indicated dose of carfilzomib at the indicated times (bottomarrows).

FIG. 11 is a graph showing the cell surface density of CD38 in multiplemyeloma cell lines.

FIG. 12 is a graph showing that hu38SB19, as the sole active ingredient,results in dose-dependent anti-tumor effects and eradication of NCI-H929hind-flank xenograft tumor growth. Four cumulative doses, given twiceweekly at 5 mg/kg were sufficient to eliminate palpable tumors in allmice within the cohort.

FIG. 13 is a graph showing that low-dose combinations of carfilzomib andhu38SB19 results in near complete tumor growth inhibition of NCI-H929xenografts.

SUMMARY OF THE INVENTION

In some embodiments, the present invention relates to a method oftreating a cancer in a subject which comprises administering one or moreanti-CD38 antibodies and one or more carfilzomib compounds to thesubject. In some embodiments, the cancer is a hematological malignancy.In some embodiments, the cancer is multiple myeloma. In someembodiments, the cancer is a relapsed multiple myeloma or a refractorymultiple myeloma. In some embodiments, the one or more carfilzomibcompounds is carfilzomib. In some embodiments, the one or more anti-CD38antibodies are administered in an effective amount, preferably asynergistic amount. In some embodiments, the one or more anti-CD38antibodies and/or the one or more carfilzomib compounds are administeredin a therapeutically effective amount. In some embodiments, at least oneof the one or more anti-CD38 antibodies is capable of killing a CD38+cell by apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC),and complement-dependent cytotoxicity (CDC). In some embodiments, theantibody is hu38SB19. In some embodiments, at least one of the one ormore anti-CD38 antibodies comprises one or morecomplementarity-determining region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 14, 81, 15, 16,17, 18, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36. In some embodiments,at least one of the one or more anti-CD38 antibodies is selected fromthe group consisting of: a) an antibody comprising a heavy chaincomprising three sequential CDRs having amino acid sequences consistingof SEQ ID NOs: 13, 15 and either SEQ ID NO: 14 or SEQ ID NO: 81, and alight chain comprising three sequential CDRs having amino acid sequencesconsisting of SEQ ID NOs: 16, 17 and 18; b) an antibody comprising aheavy chain comprising three sequential CDRs having amino acid sequencesconsisting of SEQ ID NOs: 25, 26 and 27, and a light chain comprisingthree sequential CDRs having amino acid sequences consisting of SEQ IDNOs: 28, 29 and 30; c) an antibody comprising a heavy chain comprisingthree sequential CDRs having amino acid sequences consisting of SEQ IDNOs: 1, 2 and 3, and a light chain comprising three sequential CDRshaving amino acid sequences consisting of SEQ ID NOs: 4, 5 and 6; d) anantibody comprising a heavy chain comprising three sequential CDRshaving amino acid sequences consisting of SEQ ID NOs: 7, 8 and 9, and alight chain comprising three sequential CDRs having amino acid sequencesconsisting of SEQ ID NOs: 10, 11 and 12; e) an antibody comprising aheavy chain comprising three sequential CDRs having amino acid sequencesconsisting of SEQ ID NOs: 19, 20 and 21, and a light chain comprisingthree sequential CDRs having amino acid sequences consisting of SEQ IDNOs: 22, 23 and 24; and f) an antibody comprising a heavy chaincomprising three sequential CDRs having amino acid sequences consistingof SEQ ID NOs: 31, 32 and 33, and a light chain comprising threesequential CDRs having amino acid sequences consisting of SEQ ID NOs:34, 35 and 36. In some embodiments, the antibody comprises a heavy chainhaving a VH variable region represented by SEQ ID NO: 66, and a lightchain having a VL variable region represented by either SEQ ID NO: 62 orSEQ ID NO: 64. In some embodiments, the antibody comprises a heavy chainhaving a VH variable region represented by SEQ ID NO: 72, and a lightchain having a VL variable region represented by either SEQ ID NO: 68 orSEQ ID NO: 70. In some embodiments, the one or more anti-CD38 antibodiesare administered intravenously. In some embodiments, the one or morecarfilzomib compounds are administered orally. In some embodiments, theone or more anti-CD38 antibodies and the one or more carfilzomibcompounds are administered sequentially. In some embodiments, the methodfurther comprises administering a dexamethasone compound, preferablydexamethasone, to the subject. In some embodiments, the dexamethasonecompound is administered orally. In some embodiments, the dexamethasonecompound is administered at a low dose. In some embodiments, the one ormore anti-CD38 antibodies, the one or more carfilzomib compounds, andthe dexamethasone compound are administered sequentially. In someembodiments, the one or more anti-CD38 antibodies and the one or morecarfilzomib compounds are administered sequentially.

In some embodiments, the present invention relates to a compositioncomprising a) at least one anti-CD38 antibody, preferably the antibodyis capable of killing a CD38+ cell by apoptosis, antibody-dependentcell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity(CDC); and b) at least one carfilzomib compound, preferably carfilzomib;and, optionally c) a dexamethasone compound, preferably dexamethasone.In some embodiments, the present invention relates to a compositioncomprising a) at least one anti-CD38 antibody; and b) at least onecarfilzomib compound; and, optionally i) a dexamethasone compound. Insome embodiments, the antibody is capable of killing a CD38+ cell byapoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC). In some embodiments, theantibody is hu38SB19. In some embodiments, the carfilzomib compound iscarfilzomib. In some embodiments, the dexamethasone compound isdexamethasone.

In some embodiments, the present invention is directed to a kitcomprising a) a first composition comprising at least one anti-CD38antibody, preferably the antibody is capable of killing a CD38+ cell byapoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC); and b) a second compositioncomprising at least one carfilzomib compound, preferably carfilzomib. Insome embodiments, the compositions in the kit are packaged forsequential administration to a subject. In some embodiments, theantibody is hu38SB19. In some embodiments, the kit further includes adexamethasone compound, preferably dexamethasone. In some embodiments,the carfilzomib compound and the dexamethasone compound are packaged forsequential administration to a subject.

In some embodiments, the present invention is directed to a kitcomprising at least one anti-CD38 antibody capable of killing a CD38+cell by apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC),and complement-dependent cytotoxicity (CDC), packaged together with alabel having one or more messages that the at least one anti-CD38antibody shall be administered in combination with carfilzomib, andoptionally with dexamethasone. In some embodiments, the antibody ishu38SB19. In some embodiments, the kit further includes a dexamethasonecompound, preferably dexamethasone. In some embodiments, the carfilzomibcompound and the dexamethasone compound are packaged for sequentialadministration to a subject.

In some embodiments, the present invention is directed to a combinationof: (i) at least one anti-CD38 antibody, preferably the antibody iscapable of killing a CD38+ cell by apoptosis, antibody-dependentcell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity(CDC); and (ii) at least one carfilzomib compound, preferablycarfilzomib; and, optionally (iii) a dexamethasone compound, preferablydexamethasone. In some embodiments, the present invention relates to acombination comprising a) at least one anti-CD38 antibody; and b) atleast one carfilzomib compound; and, optionally i) a dexamethasonecompound. In some embodiments, the antibody is capable of killing aCD38+ cell by apoptosis, antibody-dependent cell-mediated cytotoxicity(ADCC), and complement-dependent cytotoxicity (CDC). In someembodiments, the antibody is hu38SB19. In some embodiments, thecarfilzomib compound is carfilzomib. In some embodiments, thedexamethasone compound is dexamethasone. In some embodiments, thecombination is for sequential use in the treatment of a hematologicalmalignancy, preferably multiple myeloma.

In some embodiments, the present invention is directed to use of (i) atleast one anti-CD38 antibody, preferably the antibody is capable ofkilling a CD38+ cell by apoptosis, antibody-dependent cell-mediatedcytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC); and(ii) at least one carfilzomib compound, preferably carfilzomib; and,optionally (iii) a dexamethasone compound, preferably dexamethasone, forthe treatment of a hematological malignancy, preferably multiplemyeloma. In some embodiments, the present invention relates to use of a)at least one anti-CD38 antibody; and b) at least one carfilzomibcompound; and, optionally i) a dexamethasone compound, for the treatmentof a hematological malignancy, preferably multiple myeloma. In someembodiments, the antibody is capable of killing a CD38+ cell byapoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC). In some embodiments, theantibody is hu38SB19. In some embodiments, the carfilzomib compound iscarfilzomib. In some embodiments, the dexamethasone compound isdexamethasone.

In some of the various embodiments of the present invention, the subjectto be treated is mammalian. In some of the various embodiments of thepresent invention, the subject to be treated is a test animal such as amouse. In some of the various embodiments of the present invention, thesubject to be treated is human.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of treating a cancer in asubject which comprises administering one or more anti-CD38 antibodiesand one or more carfilzomib compounds to the subject. As used herein,“treat” or “treating” means to alleviate symptoms, eliminate thecausation of the symptoms either on a temporary or permanent basis, orto prevent or slow the appearance of symptoms of the named disorder orcondition. As disclosed herein, the efficacy of a carfilzomib compoundis considerably improved when administered in conjunction with one ormore anti-CD38 antibodies according to the present invention. In fact,the administration of one or more anti-CD38 antibodies which exhibit (a)the capability of killing a CD38⁺ cell by apoptosis, (b)antibody-dependent cell-mediated cytotoxicity (ADCC), and (c)complement-dependent cytotoxicity (CDC) is believed to considerablyimprove the efficacy of carfilzomib compounds in the treatment ofhematological malignancies, including MM, to a degree that isunexpectedly more than other anti-CD38 antibodies which do not exhibitall three (a)-(c) activities. Therefore, in some embodiments, the one ormore anti-CD38 antibodies are capable of (a) killing a CD38⁺ cell byapoptosis, (b) antibody-dependent cell-mediated cytotoxicity (ADCC), and(c) complement-dependent cytotoxicity (CDC). In some embodiments, theone or more anti-CD38 antibodies and/or the one or more carfilzomibcompounds are administered in a therapeutically effective amount. Asused herein, a “therapeutically effective amount” of a substance refersto an amount of that substance that results in the alleviation of one ormore symptoms, elimination of the causation of the symptoms either on atemporary or permanent basis, and/or the prevention or reduction in theappearance of symptoms of the named disorder or condition in themajority of subjects afflicted with and similarly treated for the nameddisease or disorder.

In some embodiments, the cancer is one in which CD38 is expressed by themalignant cells. In some embodiments, the cancer is a hematologicalmalignancy of the blood, bone marrow, and/or lymph nodes. In someembodiments, the cancer is a blood cancer. Blood cancers includemyeloma, lymphoma and leukemia. The blood cancer might, for instance, beselected from the group consisting of multiple myeloma, non-Hodgkin'slymphoma, Hodgkin's lymphoma, hairy cell leukemia, chronic lymphocyticleukemia, chronic myeloid leukemia, acute myeloid leukemia, and acutelymphocytic leukemia. In some embodiments, the cancer is multiplemyeloma (MM). In some embodiments, the cancer is a relapse MM orrefractory MM. As used herein, relapsed MM refers to clinically activeMM after a period of remission and refractory MM refers to progressiveor stable disease while being treated or progressive disease within 3months of the last does of the prior treatment. See Dimopoulos et al.(2010) Eur J Haematology 88:1-15.

In some embodiments, the subject is mammalian, preferably human. In someembodiments, the subject is an adult human, e.g., at least 18 years. Insome embodiments, the subject is in need of treatment for the cancer. Insome embodiments, the subject has been diagnosed as having the cancer.In some embodiments, the cancer is in partial or complete remission,however, the one or more carfilzomib compounds and the one or moreanti-CD38 antibodies are administered to the subject so as to reduce thelikelihood of relapse. In some embodiments, the subject has a Karnofskyperformance status equal or superior to 60%. The Karnofsky status runsfrom 100 to 0, where 100 is “perfect” health and 0 is death (Karnofskyand Burchenal, 1949, “The Clinical Evaluation of Chemotherapeutic Agentsin Cancer.” In: MacLeod C M (Ed), Evaluation of Chemotherapeutic Agents.Columbia Univ Press). In some embodiments, the subject has undergone atleast one or two prior therapies for multiple myeloma, induction therapybeing considered one prior therapy. In some embodiments, the subjectexhibits evidence that either the cancer progressed while the subjectunderwent a prior therapy, or that the subject was refractory to theprior therapy.

In some embodiments, the anti-CD38 antibodies specifically bind CD38. Insome embodiments, the anti-CD38 antibodies are raised against CD38 or anepitope thereof. In some embodiments, the anti-CD38 antibodies aremonoclonal antibodies. In some embodiments, one or more of the anti-CD38antibodies according to the present invention are monoclonal antibodiesas described in WO 2008/047242, which is herein incorporated byreference in its entirety. In some embodiments, one or more of theanti-CD38 antibodies are monoclonal antibodies 38SB13, 38SB18, 38SB19,38SB30, 38SB31, and 38SB39 as described in WO 2008/047242, which isherein incorporated by reference in its entirety. In some embodiments,the one or more anti-CD38 antibodies are capable of killing CD38⁺ cellsby three different cytotoxic mechanisms, induction of apoptosis,antibody-dependent cell-mediated cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC).

The term “antibody” is used herein in the broadest sense and includesmonoclonal antibodies (including full length monoclonal antibodies) ofany isotype such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies,multispecific antibodies, chimeric antibodies, and antibody fragments.As used herein, the prefix “anti-” when in conjunction with an antigen,indicates that the given antibody is reactive with the given antigen. Anantibody reactive with a specific antigen can be generated by syntheticand/or recombinant methods such as selection of libraries of recombinantantibodies in phage or similar vectors, or by immunizing an animal withthe antigen or an antigen-encoding nucleic acid.

A typical IgG antibody is comprised of two identical heavy chains andtwo identical light chains that are joined by disulfide bonds. Eachheavy and light chain contains a constant region and a variable region.Each variable region contains three segments called“complementarity-determining regions” (“CDRs”) or “hypervariableregions”, which are primarily responsible for binding an epitope of anantigen. They are usually referred to as CDR1, CDR2, and CDR3, numberedsequentially from the N-terminus. The more highly conserved portions ofthe variable regions outside of the CDRs are called the “frameworkregions”. As used herein, “V_(H)” or “VH” refers to the variable regionof an immunoglobulin heavy chain of an antibody, including the heavychain of an Fv, scFv, dsFv, Fab, Fab′ or F(ab′)₂ fragment. Reference to“V_(L)” or “VL” refers to the variable region of the immunoglobulinlight chain of an antibody, including the light chain of an Fv, scFv,dsFv, Fab, Fab′ or F(ab′)₂ fragment.

The antibodies according to the present invention may be, e.g., murine,chimeric, and/or humanized antibodies. As used herein, a “chimericantibody” is an antibody in which the constant region, or a portionthereof, is altered, replaced, or exchanged, so that the variable regionis linked to a constant region of a different species, or belonging toanother antibody class or subclass. “Chimeric antibody” also refers toan antibody in which the variable region, or a portion thereof, isaltered, replaced, or exchanged, so that the constant region is linkedto a variable region of a different species, or belonging to anotherantibody class or subclass. Methods for producing chimeric antibodiesare known in the art. See e.g., Morrison, 1985, Science, 229: 1202; Oiet al., 1986, BioTechniques, 4: 214; Gillies et al., 1989, J. Immunol.Methods, 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397, which are incorporated herein by reference in theirentireties. The term “humanized antibody”, as used herein, refers to achimeric antibody which contain minimal sequence derived from non-humanimmunoglobulin. The goal of humanization is a reduction in theimmunogenicity of a xenogenic antibody, such as a murine antibody, forintroduction into a human, while maintaining the full antigen bindingaffinity and specificity of the antibody. Humanized antibodies, orantibodies adapted for non-rejection by other mammals, may be producedusing several technologies such as resurfacing and CDR grafting. As usedherein, the resurfacing technology uses a combination of molecularmodelling, statistical analysis and mutagenesis to alter the non-CDRsurfaces of antibody variable regions to resemble the surfaces of knownantibodies of the target host. The CDR grafting technology involvessubstituting the complementarity determining regions of, for example, amouse antibody, into a human framework domain, e.g., see WO 92/22653.

Humanized chimeric antibodies preferably have constant regions andvariable regions other than the complementarity determining regions(CDRs) derived substantially or exclusively from the corresponding humanantibody regions and CDRs derived substantially or exclusively from amammal other than a human.

Strategies and methods for the resurfacing of antibodies, and othermethods for reducing immunogenicity of antibodies within a differenthost, are disclosed in U.S. Pat. No. 5,639,641, which is herebyincorporated in its entirety by reference. Antibodies can be humanizedusing a variety of other techniques including CDR-grafting (EP 0 239400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089), veneering orresurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991, MolecularImmunology 28(4/5): 489-498; Studnicka G. M. et al., 1994, ProteinEngineering, 7(6): 805-814; Roguska M. A. et al., 1994, PNAS, 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and identificationof flexible residues (PCT/US2008/074381). Human antibodies can be madeby a variety of methods known in the art including phage displaymethods. See also U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and5,814,318; and international patent application publication numbers WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741 (said references incorporated by reference intheir entireties).

In some embodiments, one or more of the anti-CD38 antibodies accordingto the invention are capable of killing a CD38⁺ cell by apoptosis,antibody-dependent cell-mediated cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC). In some embodiments, one ormore of the anti-CD38 antibodies according to the invention are capableof killing said CD38⁺ cells by apoptosis even in the absence of stromacells or stroma-derived cytokines. These activities can be assessed asdescribed in WO 2008/047242, which is hereby incorporated by referencein its entirety.

In some embodiments according to the invention, one or more anti-CD38antibodies are selected from the group consisting of 38SB13, 38SB18,38SB19, 38SB30, 38SB31, 38SB39, and antibodies cross-competing with38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39. The hybridoma celllines producing the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39murine anti-CD38 antibodies have been deposited at the American TypeCulture Collection (10801 University Bld, Manassas, Va., 20110-2209,USA), on 21 Jun. 21 2006, under the deposit numbers PTA-7667, PTA-7669,PTA-7670, PTA-7666, PTA-7668, and PTA-7671, respectively (as describedin WO 2008/047242, which is herein incorporated by reference in itsentirety).

As disclosed herein, references to SEQ ID NOs refers to the sequencesset forth in the Sequence Listing submitted herewith and also as recitedin WO 2008/047242, which is herein incorporated by reference in itsentirety. In some embodiments, the anti-CD38 antibodies according to thepresent invention may, for instance, comprise a heavy chain comprisingthree sequential CDRs having amino acid sequences represented by SEQ IDNOs: 1, 2, and 3, and a light chain comprising three sequential CDRshaving amino acid sequences represented by SEQ ID NOs: 4, 5, and 6. Anexample of such an antibody is the 38SB13 antibody, which comprises aheavy chain having a V_(H) variable region represented by SEQ ID NO: 50,and a light chain having a V_(L) variable region represented by SEQ IDNO: 38.

In some embodiments, the anti-CD38 antibodies according to the presentinvention may, for instance, comprise a heavy chain comprising threesequential CDRs having amino acid sequences represented by SEQ ID NOs:7, 8, and 9, and a light chain comprising three sequential CDRs havingamino acid sequences represented by SEQ ID NOs: 10, 11, and 12. Anexample of such an antibody is the 38SB18 antibody, which comprises aheavy chain having a V_(H) variable region represented by SEQ ID NO: 52and a light chain having a V_(L) variable region represented by SEQ IDNO: 40.

In some embodiments, the anti-CD38 antibodies according to the presentinvention may, for instance, comprise a heavy chain comprising threesequential CDRs having amino acid sequences represented by SEQ ID NO:13, SEQ ID NO: 15 and either SEQ ID NO: 14 or SEQ ID NO: 81, and a lightchain comprising three sequential CDRs having amino acid sequencesrepresented by SEQ ID NOs: 16, 17, and 18. An example of such anantibody is the 38SB19 antibody, which comprises a heavy chain having aV_(H) variable region represented by SEQ ID NO: 54 and a light chainhaving a V_(L) variable region represented by SEQ ID NO: 42. Specificexamples of humanized versions of 38SB19 (hu38SB19) include antibodiescomprising a heavy chain having a V_(H) variable region represented bySEQ ID NO: 66, and a light chain having a V_(L) variable regionrepresented by either SEQ ID NO: 62 or SEQ ID NO: 64. hu38SB19 is ahumanized anti-CD38 antibody currently undergoing clinical evaluation inCD38-positive hematologic malignancies, including multiple myeloma.Previous and current studies demonstrate that the anti-myeloma activityassociated with this agent involve mechanisms of ADCC, and CDC, as wellas novel, direct apoptotic and anti-ADP-ribosyl cyclase activity. SeeMarie-Cécile Wetzel, Céline Nicolazzi, François Vallée, et al. hu38SB19:characterization of a potent phase I humanized anti-CD38 antibody forthe treatment of multiple myeloma and other hematologic malignancies.AACR Annual Meeting 2013, Abstract #4735.

In some embodiments, the anti-CD38 antibodies according to the presentinvention may, for instance, comprise a heavy chain comprising threesequential CDRs having amino acid sequences represented by SEQ ID NOs:19, 20, and 21, and a light chain comprising three sequential CDRshaving amino acid sequences represented by SEQ ID NOs: 22, 23, and 24.An example of such an antibody is the 38SB30 antibody, which comprises aheavy chain having a V_(H) variable region represented by SEQ ID NO: 56and a light chain having a V_(L) variable region represented by SEQ IDNO: 44.

In some embodiments, the anti-CD38 antibodies according to the presentinvention may, for instance, comprise a heavy chain comprising threesequential CDRs having amino acid sequences represented by SEQ ID NOs:25, 26, and 27, and a light chain comprising three sequential CDRshaving amino acid sequences represented by SEQ ID NOs: 28, 29, and 30.An example of such an antibody is the 38SB31 antibody, which comprises aheavy chain having a V_(H) variable region represented by SEQ ID NO: 58and a light chain having a V_(L) variable region represented by SEQ IDNO: 46. Specific examples of humanized versions of 38SB31 (hu38SB31)include antibodies comprising a heavy chain having a V_(H) variableregion represented by SEQ ID NO: 72, and a light chain having a V_(L)variable region represented by either SEQ ID NO: 68 or SEQ ID NO: 70.

In some embodiments, the anti-CD38 antibodies according to the presentinvention may, for instance, comprise a heavy chain comprising threesequential CDRs having amino acid sequences represented by SEQ ID NOs:31, 32 and 33, and a light chain comprising three sequential CDRs havingamino acid sequences represented by SEQ ID NOs: 34, 35, and 36. Anexample of such an antibody is the 38SB39 antibody, which comprises aheavy chain having a V_(H) variable region represented by SEQ ID NO: 60and a light chain having a V_(L) variable region represented by SEQ IDNO: 48.

In some embodiments, the anti-CD38 antibodies according to the inventionare humanized antibodies consisting of two identical heavy chains and oftwo identical light chains, wherein each chain consists of one constantregion and of one variable region.

As used herein, a “carfilzomib compound” refers to carfilzomib(S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamideand carfilzomib derivatives. As used herein, “carfilzomib derivatives”refers to compounds which have2-acetamido-N-(1-((1-(1-methylcyclopropyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide,i.e.,

which may or may not be substituted, as part of its structural formula.In some embodiments, carfilzomib derivatives include compounds whichhave the following structure, which may or may not be substituted, aspart of its structural backbone:

wherein X is selected from O, NH, and N—C₁₋₆alkyl, preferably O.Examples of “carfilzomib derivatives” according to the present inventioninclude those as set forth in U.S. Pat. Nos. 7,232,818; 7,417,042;7,491,704; 7,737,112; 8,129,346; 8,207,125; 8,207,126; 8,207,127; and8,207,297.

In some embodiments, the one or more anti-CD38 antibodies areadministered in an effective amount. As used herein, an effective amountof the one or more anti-CD38 antibodies is an amount which results in anadditive or a synergistic effect with the one or more carfilzomibcompounds. As used herein, a “synergistic amount” is one that results ina synergistic effect. As used herein, a “synergistic effect” refers tothe effect of the combination of the one or more anti-CD38 antibodiesand the one or more carfilzomib compounds which is more than theirexpected additive effect. In some embodiments, the one or more anti-CD38antibodies are administered before, during, and/or after theadministration of the one or more carfilzomib compounds. In someembodiments, the one or more anti-CD38 antibodies and the one or morecarfilzomib compounds are co-administered in the form of a singlecomposition, e.g., as a mixture.

Thus, in some embodiments, the present invention is directed tocompositions comprising a mixture of at least one anti-CD38 antibody andat least one carfilzomib compound. In some embodiments, the mixturecomprises the at least one anti-CD38 antibody in an amount that resultsin an additive or a synergistic effect with the at least one carfilzomibcompound in a subject when both are administered. In some embodiments,the at least one anti-CD38 antibody in the mixture is one which iscapable of killing a CD38⁺ cell by apoptosis, antibody-dependentcell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity(CDC); and at least one carfilzomib compound.

For the purposes of the present invention, the methods and compositionsof the present invention are not exclusively limited to those which areobtained by physical association of the anti-CD38 antibodies and thecarfilzomib compound, but also to those which permit a separateadministration, which can be simultaneous or spaced out over a period oftime. Thus, in some embodiments, the present invention is directed to afirst composition comprising the one or more anti-CD38 antibodies, and asecond composition comprising one or more carfilzomib compounds. In someembodiments, the at least one anti-CD38 antibody is one which is capableof killing a CD38⁺ cell by apoptosis, antibody-dependent cell-mediatedcytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC); and atleast one carfilzomib compound. In some embodiments, the amount of theone or more anti-CD38 antibodies provided in the first composition isone that results in an additive or a synergistic effect with the atleast one carfilzomib compound in the second composition in a subjectwhen both are administered.

In some embodiments, the first and second compositions may be packagedin a kit. Thus, in some embodiments, the present invention is directedto kits which comprise a first composition comprising the one or moreanti-CD38 antibodies, and a second composition comprising one or morecarfilzomib compounds. In some embodiments, the first and secondcomposition may be mixed together before administering to a subject. Insome embodiments, the first and second compositions, may be administeredeither simultaneously or sequentially (i.e., spaced out over a period oftime) so as to obtain the maximum efficacy, additivity, synergy, or acombination thereof of the combination. In some embodiments, the presentinvention is directed to kits comprising at least one anti-CD38 antibodypackaged together with a label having one or more messages that theanti-CD38 antibody shall or might be administered in combination withcarfilzomib and optionally with dexamethasone. The kits according to thepresent invention may further comprise one or more messages that theantibody shall or might be administered to a subject suffering from ablood cancer such as multiple myeloma (e.g., relapsed or refractorymultiple myeloma). In some embodiments, the one or more anti-CD38antibodies in the kits of the present invention are those which arecapable of killing a CD38⁺ cell by apoptosis, antibody-dependentcell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity(CDC).

In some embodiments, the compositions of the present invention arepharmaceutical compositions. As used herein, the term “pharmaceuticalcomposition” refers to a composition comprising at least one activeprinciple (e.g., an anti-CD38 antibody or a carfilzomib compound) and atleast one pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well known to the skilled in the art, andusually depend on the chosen route of administration. Pharmaceuticalcompositions according to the present invention may be provided in anyform or formulation that is suitable for the chosen route ofadministration, such as e.g., a solution in case of an intravenous routeof administration, e.g., capsules, pills or tablets in case of an oralroute of administration, etc.

The dosage regimen of the active principles and of the pharmaceuticalcomposition described herein can be chosen by prescribing physicians,based on their knowledge of the art, including information published byregulatory authorities. For example, carfilzomib is typicallyadministered intravenously. According to the U.S. Food and DrugAdministration (FDA), carfilzomib might be administered intravenously,e.g., over 2 to 10 minutes, on two consecutive days each week for threeweeks (Days 1, 2, 8, 9, 15, and 16), followed by a 12-day rest period(Days 17 to 28). In some embodiments, the recommended Cycle 1 dose is 20mg/m²/day and, if tolerated, the doses of Cycle 2 and subsequent cyclesare increased to 27 mg/m²/day. In some embodiments, patients arehydrated prior to and/or following administration. Since, however,co-administration of the one or more anti-CD38 antibodies and the one ormore carfilzomib compounds results in an additive or a synergisticeffect, the dosing of the carfilzomib compound may be adjustedaccordingly, e.g., the dose changed and/or the dosing schedule modified.Of course, prescribing physicians might reconsider which dose andschedule to use depending on the condition and disease status of thepatient and based upon clinical and laboratory findings.

As the FDA recommends pre-medication with dexamethasone prior to allCycle 1 doses, during the first cycle of dose escalation, and ifinfusion reaction symptoms develop or reappear, the methods andcompositions of the present invention may further include dexamethasone,which is member of the glucocorticoid class of steroid drugs, and actsas an anti-inflammatory and immunosuppressant. Thus, in someembodiments, the treatment methods of the present invention furthercomprise administering a dexamethasone compound to the subject beingtreated with the one or more anti-CD38 antibodies and the one or morecarfilzomib compounds. Similarly, the compositions and kits of thepresent invention which comprise the one or more anti-CD38 antibodiesand/or the one or more carfilzomib compounds may further comprise adexamethasone compound. As used herein, a “dexamethasone compound”refers to dexamethasone((8S,9R,10S,11S,13S,14S,16R,17R)-9-Fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one)and dexamethasone derivatives. As used herein, a “dexamethasonederivative” refers to a compound having the following structuralformula:

wherein R1-R17 are each independently H, a halogen, an alkyl, an alkoxy,amino, or an alkylamine. In some preferred embodiments, R1-R3 are H. Insome preferred embodiments, R4-R6 are methyl. In some preferredembodiments, R7 is a halogen, preferably fluorine. In some preferredembodiments, R8 is H. In some preferred embodiments, R1-R3 are H, R4-R6are methyl, R7 is a halogen, preferably fluorine, and R8 is H.

In some embodiments, the dexamethasone compound may be administeredorally. In some embodiments, the dexamethasone compound may beadministered at the same or a lower dose than the dose recommended fordexamethasone by the EMA.

The compositions of the present invention may be used as a medicamentand/or for use in the manufacture of a medicament. In some embodiments,the compositions of the present invention may be used as a medicamentand/or for use in the manufacture of a medicament for use in thetreatment of a cancer such as a hematological malignancy of the blood,bone marrow, and/or lymph nodes, preferably a blood cancer.

Several documents are cited throughout the text of this specification.Each of the documents herein (including any journal article or abstract,published or unpublished patent application, issued patent,manufacturer's specifications, instructions, etc.) are herebyincorporated by reference. However, there is no admission that anydocument cited herein is indeed prior art in respect of the presentinvention.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLES

hu38SB19 was provided in solution at 5 mg/ml, stored at 4° C. It wasdiluted into sterile saline in preparation for dosing, stored at 4° C.and used within 10 days of dilution.

Carfilzomib (PR-171) was obtained from Chemie Tek (CT-CARF 98).Carfilzomib was formulated in an aqueous solution of 10% (w/v)sulfobutylether-h-cyclodextrin (Cydex) and 10 mmol/L sodium citrate (pH3.5), 2 mg/ml stock prepared and frozen at −80° C., diluted daily withvehicle before injection. Carfilzomib was administered weekly qd×2×3 wk(iv).

Example 1 Effect of the Administration of Both Anti-CD38 Antibody andCarfilzomib in a Mice Model of MM

These studies under this Example were done under approval of the UCSFIACUC.

The subcutaneous multiple myeloma (MM) xenograft mouse models wereestablished using NCI-H929 or RPMI-8226 cell lines. Specifically, 5-6week old female Balb/c Scid mice were obtained from Jackson Lab. Micewere housed for 7-10 days prior to implantation. Mice were housed in adedicated room in the UCSF Mt Zion Animal Barrier Facility. NCI-H929 andRPMI-8226 cells were obtained from the German Collection ofMicroorganisms and Cell Cultures, DSMZ, (Deutsche Sammlung vonMikroorganismen and Zellkulturen), and grown in sterile suspensionculture in T225 flasks as follows: NCI-H929: RPMI1640+20% FBS+4 mML-glutamine+1 mM sodium pyruvate+50 μM mercaptoethanol. RPMI-8226:RPMI1640+10% FBS+4 mM L-glutamine.

At the time of implantation, mice were shaved on the right flank andshoulder region and anesthetized with ip avertin. MM cells suspended inserum free RPMI 1640 media diluted 1:1 with Matrigel (BD) at aconcentration of 1×10⁸ cells per ml were injected sc into the rightflank in 100 μL volume (1×10⁷ cells) using a 1 ml syringe and 25 gneedle. Mice were monitored twice weekly for the appearance of tumorsand once tumors were visible, measurements were collected twice weeklyfor body weight and tumor volume. Electronic balance and calipers wereused and data was collected directly into a study management program(Study Director). When the mean tumor volume reached about 150-200 mm³,the mice were distributed into treatment groups of 8-10 mice per groupsand dosing was begun.

The dosing schedule was hu38SB19 was 2×/wk×2 wk (iv, lateral tail vein)and carfilzomib was weekly qd×2×3 wk (iv, lateral tail vein) (once perday, two days a week for three weeks). Dose levels for use incombination studies are as follows:

Cell Type Carfilzomib hu38SB19 NCI-H929  2 mpk 0.5 mpk RPMI 8226 2.5 mpk 15 mpk mpk = mg per kg body weight

Data were collected using electronic balance and calipers using a studymanagement application called StudyLog (Study Director). Graphs aretaken directly from the application. The experimental results areprovided in FIGS. 1A-10B.

Based on the single agent results of hu38SB19 and carfilzomib inRPMI-8226 and NCI-H929 multiple myeloma xenograft models, the H929 modelappears to be a more sensitive model to both agents while the RPMI modelseems to be more resistant to the treatments even at the highest dosestested (FIGS. 1A-7B). Therefore in the combination studies, a suboptimaldose for each agent was chosen to evaluate the activity of thecombination treatment (carfilzomib+hu38SB19) in the H929 model whilehigher doses of carfilzomib and hu38SB19 were tested in the RPMI model.

Antitumor activity was determined according to NCI standards based onthe ratio of the median tumor volume change of the treated/median tumorvolume change of the control×100 (%ΔT/ΔC). Low numerical values forΔT/ΔC describe stronger anti-tumor activity. Anti-tumor activity isdefined as T/C<40% at minimum. ΔT/ΔC<10% is considered high anti-tumoractivity.

In the H929 model, hu38SB19 alone at 0.5 mg/kg/injection (twice a weekfor 2 weeks) was inactive with a %ΔT/ΔC of 74%. Treatment withcarfilzomib alone at 2 mg/kg (twice a week for three weeks) was inactive(68%ΔTΔ/C). The combination of hu38SB19 (0.5 mg/kg/injection) andcarfilzomib (2 mg/kg/injection) had much higher activity (tumorregression) with %ΔT/ΔC of −11% (FIG. 8). The results are summarized inTable 1.

TABLE 1 Anti-tumor efficacy of hu38SB19 in combination with carfilzomibagainst NCI-H929 multiple myeloma model Schedule of Dose in mg/kgAdministration % ΔT/ΔC Agent (total dose) IV route (D69) Activity PBS —2x/wk x 2 wk (IV) hu38SB19 0.5 (2) 2x/wk x 2 wk (IV) 74 InactiveCarfilzomib   2 (12) 2x/wk x 3 wk (IV) 68 Inactive hu38SB19 + 0.5 (2) +2x/wk x 2 wk (IV) + −11 Highly Carfilzomib 2 (12) 2x/wk x 3 wk (IV)Active % ΔT/ΔC Median tumor volume change of the treated/Median tumorvolume change of the control x 100, IV = intravenous, wk = week, PBS:phosphate buffered saline

As shown in FIGS. 10A-10B, similar results were obtained in theRPMI-8226 xenograft models. In particular, on Day 41, carfilzomib (3mg/kg qd×2 every wk×3 wk) resulted in 0/10 complete regressions;hu38SB19 (3 mg/kg BIW×2 wk) resulted in 2/10 complete regressions. Thus,the additive expectation based on extrapolation for the combination ofcarfilzomib and hu38SB19 would be expected to be 2/10 completeregressions. However, the combination of carfilzomib and hu38SB19surprisingly resulted in 5/8 complete regressions which is more than 3times the expected result.

In both the NCI-H929 and RPMI-8226 xenograft models, the combinationtreatment inhibited tumor growth to a much greater extent than a singleagent alone, indicating the combination of hu38SB19 and carfilzomibblocked tumor cell growth through potential synergistic mechanisms.Carfilzomib is a second generation proteasome inhibitor which wasrecently approved to treat relapsed and refractory multiple myelomapatients. Inhibition of proteasome activity by carfilzomib results in abuild-up of polyubiquinated proteins, which may cause cell cycle arrest,apoptosis, and inhibition of tumor growth. Hu38SB19 has demonstratedmultiple mechanisms of action including ADCC, CDC, and direct apoptosisinduction.

It has been reported that some CD38 antibodies such as Daratumumab isable to induce apoptosis only after cross-linking with a secondaryantibody without much direct effect by itself. However, in preclinicalstudies, hu38SB19 demonstrated potent direct pro-apoptotic activity ontumor cells without cross-linking. Thus, this unique property ofhu38SB19 may also lead to greater tumor cell killing when in combinationwith carfilzomib compared to other CD38 antibodies combined withcarfilzomib.

Example 2 Effect of the Administration of Both Anti-CD38 Antibody andCarfilzomib in Humans

A clinical study for evaluating the effects of a treatment with hu38SB19combined with carfilzomib in patients with relapsed or refractorymultiple myeloma may be performed as described below.

The goals of the study may include:

-   -   To determine the efficacy and the maximum tolerated dose;    -   To evaluate the safety, including immunogenicity, of hu38SB19 in        combination with carfilzomib in relapse or refractory multiple        myeloma. The severity, frequency and incidence of all toxicities        is assessed;    -   To evaluate the pharmacokinetics (PK) of hu38SB19 when        administered in combination with carfilzomib and the PK of        carfilzomib in combination with hu38SB19, and optionally        dexamethasone.    -   To assess the relationship between clinical (adverse event        and/or tumor response) effects and pharmacologic parameters        (PK/pharmacodynamics), and/or biologic (correlative laboratory)        results;    -   Estimate the activity (response rate) using International        Myeloma Working Group defined response criteria of hu38SB19 plus        carfilzomib, and optionally dexamethasone; and    -   To describe overall survival, progression free survival (PFS)        and time to disease progression in patients treated with this        combination.

Patients with relapsed multiple myeloma who have received at least twoprior treatments (including bortezomib and thalidomide and/orlenalidomide) and whose disease has a less than or equal to 25% responseto the most recent therapy or has disease progression during or within60 days of the most recent therapy are enrolled. Patients excluded fromthe trial are those having total bilirubin levels ≧2× upper limit ofnormal (ULN); creatinine clearance rates <30 mL/min; New York HeartAssociation Class III to IV congestive heart failure; symptomaticcardiac ischemia; myocardial infarction within the last 6 months;peripheral neuropathy Grade 3 or 4, or peripheral neuropathy Grade 2with pain; active infections requiring treatment; and pleural effusion.

Carfilzomib is administered intravenously over 2 to 10 minutes on twoconsecutive days each week for three weeks, followed by a 12-day restperiod (28-day treatment cycle), until disease progression, unacceptabletoxicity, or for a maximum of 12 cycles. Patients receive 20 mg/m² ateach dose in Cycle 1, and 27 mg/m² in subsequent cycles. To reduce theincidence and severity of fever, rigors, chills, dyspnea, myalgia, andarthralgia, dexamethasone 4 mg by mouth or by intravenous infusion maybe administered prior to all carfilzomib doses during the first cycleand prior to all carfilzomib doses during the first dose-escalation (27mg/m²) cycle. Dexamethasone premedication (4 mg orally or intravenously)may be reinstated if these symptoms reappeared during subsequent cycles.Doses of hu38SB19 may be administered on the same days the carfilzomibdoses are administered and/or on different days. When administered onthe same days, hu38SB19 and carfilzomib may be administered at the sametime as one composition or as two separate compositions.

The study duration for an individual patient includes a screening periodfor inclusion of up to 21 days, and at least 4 weeks of treatment in theabsence of severe adverse reaction, dose limiting toxicity or diseaseprogression plus up to 60 days post-treatment follow up. The totalduration of the study may be up to one year.

The following parameters may be measured during and/or at the end of thestudy:

-   -   Number of patients with adverse events when treated with        hu38SB19 in combination with carfilzomib;    -   Assessment of partial response, complete response, progression        free survival, and survival;    -   Assessment of the following PK parameters: area under curve        (AUC), maximum concentration (Cmax) and plasma half-life (T ½);    -   Number of CD38 receptors occupied by hu38SB19; and    -   Number of anti-SAR antibodies in response to hu38SB19.

Example 3 Efficacy of Anti-CD38 Antibody in In Vivo Tumor Models ofMultiple Myeloma as a Single-Agent or in Combination with andCarfilzomib A. Materials and Methods

CD38 Density: CD38 density was determined using anti-CD38-PE Quantibrite(BD Biosciences; Cat.342371) per the manufacturer's recommendedprotocols.

Reagents & Compounds: hu38SB19 was provided by Sanofi Oncology insolution at 5 mg/ml and stored at 4° C. hu38SB19 was diluted intosterile saline in preparation for dosing and used within 10 days ofdilution. hu38SB19 was administered twice weekly×2 wk IV. Carfilzomib(PR-171) was obtained from Chemie Tek (CT-CARF 98). Carfilzomib wasformulated in an aqueous solution of 10% (w/v)sulfobutylether-h-cyclodextrin (Cydex) and 10 mmol/L sodium citrate (pH3.5), 2 mg/ml stock prepared and frozen at −80° C., diluted daily withvehicle before injection. Carfilzomib was administered weekly qd×2×3 wk(iv).

Test Animals: 5-6 week old female Balb/c Scid mice were obtained fromJackson Lab. Mice were housed for 7-10 days prior to implantation ofmultiple myeloma (MM) cell lines. Mice were housed in a dedicated roomin the UCSF Mt. Zion Animal Barrier Facility.

Xenograft Model: At the time of implantation, mice were shaved on theright flank and shoulder. MM cells were suspended in serum free RPMI1640 media diluted 1:1 with Matrigel (BD) at a concentration of 1×10⁸cells per ml were injected sc into the right flank in 100 ul volume(1×10⁷ cells) using a 1 ml syringe and 25 g needle. Mice were monitoredtwice weekly for the appearance of tumors and once tumors were visible,measurements were collected twice weekly for body weight and tumorvolume. Electronic balance and calipers were used and data was collecteddirectly into a study management program (Study Director). When the meantumor volume reached approximately 150-200 mm³, mice were distributedinto treatment groups of 8-10 mice per group and dosing was initiated.

B. Summary and Conclusions

hu38SB19 is a humanized anti-CD38 antibody whose anti-myeloma effectsincorporate mechanisms of ADCC, CDC, and direct apoptosis. FIG. 11 showsthe cell surface density of CD38 in multiple myeloma cell lines. See KimD, Park C Y, Medeiros B C, Weissman I L. CD19-CD45 low/−CD38 high/CD138+plasma cells enrich for human tumorigenic myeloma cells. Leukemia. 2012December, 26(12):2530-7. CD38-positive multiple myeloma plasma cellsdemonstrate variable CD38 cell surface densities. All cell lines, withthe exception of XG-6, are reported as CD38-positive. See Bataille R,Jégo G, Robillard N, et al. The phenotype of normal, reactive andmalignant plasma cells. Identification of “many and multiple myelomas”and of new targets for myeloma therapy. Haematologica. 2006 September,91(9):1234-40. Binding of hu38SB19 to CD38 also impinges on the ADPRCenzymatic activity of CD38. In vivo, hu38SB19 demonstrates potentanti-tumor effects in multiple myeloma xenografts, a disease largelycharacterized by neoplastic plasma cells expressing CD38. FIG. 12 showsthat single-agent administration of hu38SB19 results in dose-dependentinhibition of tumor growth in an NCI-H929 hind-flank model. Themagnitude and significance of tumor growth inhibition at the end of thestudy increased with increased doses of hu38SB19. FIG. 13 shows that acombined regimen of hu38SB19 and carfilzomib results in significanttumor growth inhibition in an NCI-H929 xenograft model that is notrobustly sensitive to single-agent therapy with carfilzomib. These datademonstrate that single-agent hu38SB19 inhibits growth of NCI-H929tumors and combines with sub-efficacious doses of carfilzomib to producesignificant inhibition of tumor growth. Taken together, these datasupport further evaluation of hu38SB19, both as a single-agent and incombination with standard-of-care treatment regimens, as a potentialtherapy for the treatment of multiple myeloma.

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations, andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

What is claimed is:
 1. A method of treating a cancer in a subject whichcomprises administering one or more anti-CD38 antibodies and one or morecarfilzomib compounds to the subject.
 2. The method of claim 1, whereinthe cancer is a hematological malignancy.
 3. The method of claim 1,wherein the cancer is multiple myeloma.
 4. The method of claim 1,wherein the cancer is a relapsed multiple myeloma or a refractorymultiple myeloma.
 5. The method of claim 1, wherein the one or morecarfilzomib compounds is carfilzomib.
 6. The method of claim 1, whereinthe one or more anti-CD38 antibodies are administered in an effectiveamount, preferably a synergistic amount.
 7. The method of claim 1,wherein the one or more anti-CD38 antibodies and/or the one or morecarfilzomib compounds are administered in a therapeutically effectiveamount
 8. The method of any one of claims 1 to 7, wherein at least oneof the one or more anti-CD38 antibodies is capable of killing a CD38⁺cell by apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC),and complement-dependent cytotoxicity (CDC).
 9. The method of any one ofclaims 1 to 7, wherein at least one of the one or more anti-CD38antibodies comprises one or more complementarity-determining regionhaving an amino acid sequence selected from the group consisting of SEQID NOs: 13, 14, 81, 15, 16, 17, 18, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35and
 36. 10. The method of any one of claims 1 to 7, wherein at least oneof the one or more anti-CD38 antibodies is selected from the groupconsisting of: a) an antibody comprising a heavy chain comprising threesequential CDRs having amino acid sequences consisting of SEQ ID NOs:13, 15 and either SEQ ID NO: 14 or SEQ ID NO: 81, and a light chaincomprising three sequential CDRs having amino acid sequences consistingof SEQ ID NOs: 16, 17 and 18; b) an antibody comprising a heavy chaincomprising three sequential CDRs having amino acid sequences consistingof SEQ ID NOs: 25, 26 and 27, and a light chain comprising threesequential CDRs having amino acid sequences consisting of SEQ ID NOs:28, 29 and 30; c) an antibody comprising a heavy chain comprising threesequential CDRs having amino acid sequences consisting of SEQ ID NOs: 1,2 and 3, and a light chain comprising three sequential CDRs having aminoacid sequences consisting of SEQ ID NOs: 4, 5 and 6; d) an antibodycomprising a heavy chain comprising three sequential CDRs having aminoacid sequences consisting of SEQ ID NOs: 7, 8 and 9, and a light chaincomprising three sequential CDRs having amino acid sequences consistingof SEQ ID NOs: 10, 11 and 12; e) an antibody comprising a heavy chaincomprising three sequential CDRs having amino acid sequences consistingof SEQ ID NOs: 19, 20 and 21, and a light chain comprising threesequential CDRs having amino acid sequences consisting of SEQ ID NOs:22, 23 and 24; and f) an antibody comprising a heavy chain comprisingthree sequential CDRs having amino acid sequences consisting of SEQ IDNOs: 31, 32 and 33, and a light chain comprising three sequential CDRshaving amino acid sequences consisting of SEQ ID NOs: 34, 35 and
 36. 11.The method of claim 10, wherein said antibody comprises a heavy chainhaving a V_(H) variable region represented by SEQ ID NO: 66, and a lightchain having a V_(L) variable region represented by either SEQ ID NO: 62or SEQ ID NO:
 64. 12. The method of claim 10, wherein said antibodycomprises a heavy chain having a V_(H) variable region represented bySEQ ID NO: 72, and a light chain having a V_(L) variable regionrepresented by either SEQ ID NO: 68 or SEQ ID NO:
 70. 13. The method ofany one of claims 1 to 12, wherein the one or more anti-CD38 antibodiesis administered intravenously.
 14. The method of any one of claims 1 to12, wherein the one or more carfilzomib compounds is administeredintravenously.
 15. The method of any one of claims 1 to 14, wherein theone or more anti-CD38 antibodies and the one or more carfilzomibcompounds are administered sequentially.
 16. The method of any one ofclaims 1 to 15, and further comprising administering a dexamethasonecompound, preferably dexamethasone, to the subject.
 17. The method ofclaim 16, wherein the dexamethasone compound is administered orally. 18.The method of any one of claims 16 to 17, wherein the one or moreanti-CD38 antibodies, the one or more carfilzomib compounds, and thedexamethasone compound are administered sequentially.
 19. A compositioncomprising a) at least one anti-CD38 antibody, preferably the antibodyis capable of killing a CD38⁺ cell by apoptosis, antibody-dependentcell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity(CDC); and b) at least one carfilzomib compound, preferably carfilzomib;and, optionally c) a dexamethasone compound, preferably dexamethasone.20. A kit comprising a) a first composition comprising at least oneanti-CD38 antibody, preferably the antibody is capable of killing aCD38⁻ cell by apoptosis, antibody-dependent cell-mediated cytotoxicity(ADCC), and complement-dependent cytotoxicity (CDC); and b) a secondcomposition comprising at least one carfilzomib compound, preferablycarfilzomib.
 21. The kit of claim 20, wherein the compositions arepackaged for sequential administration to a subject.
 22. The kit ofclaim 20 or claim 21, and further including a dexamethasone compound,preferably dexamethasone.
 23. The kit of claim 22, wherein thedexamethasone compound is packaged for sequential administration to asubject.
 24. A kit comprising at least one anti-CD38 antibody capable ofkilling a CD38⁺ cell by apoptosis, antibody-dependent cell-mediatedcytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC),packaged together with a label having one or more messages that the atleast one anti-CD38 antibody shall be administered in combination withcarfilzomib, and optionally with dexamethasone.
 25. A combination of:(i) at least one anti-CD38 antibody, preferably the antibody is capableof killing a CD38+ cell by apoptosis, antibody-dependent cell-mediatedcytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC); and(ii) at least one carfilzomib compound, preferably carfilzomib; and,optionally (iii) a dexamethasone compound, preferably dexamethasone. 26.The combination of claim 25, wherein the combination is for sequentialuse in the treatment of a hematological malignancy, preferably multiplemyeloma.